This invention relates to oligonucleotide analogues, their preparation and their use.
In accordance with the invention, oligonucleotide analogues can be prepared which have good hybridisation properties to single- and double-stranded nucleic acids, RNase H-activating properties, good hydrolytic stability and good stability towards cleavage by nucleases, facilitating their use as inhibitors of gene expression, for example by antisense interaction, and as pharmaceuticals in the treatment of diseases such as cancer and viruses such as influenza, herpes and HIV.
Accordingly, the present invention provides an oligonucleotide analogue having 10 to 200 natural and/or synthetic nucleoside units linked by internucleoside linkages, at least one of the internucleoside linkages being of formula 
where the indicated methylene group is attached to a 3xe2x80x2 carbon atom of a nucleoside, the indicated oxygen atom is attached to a 5xe2x80x2 carbon atom of an adjacent nucleoside, R1 is hydrogen, hydroxy, Oxe2x88x92, thiol, Sxe2x88x92, xe2x80x94NH2 or a group of formula R1a, xe2x80x94OR1a, xe2x80x94SR1a, xe2x80x94NHR1b or xe2x80x94NR1bR1c where R1a is an unsubstituted or substituted C1 to C10 alkyl, C2 to C10 alkenyl, C3 to C8 cycloalkyl, C6 to C10 aryl or C7 to C13 aralkyl group and R1b and R1c are each independently an unsubstituted or substituted C1 to C10 alkyl, C2 to C10 alkenyl, C3 to C8 cycloalkyl, C6 to C10 aryl or C7 to C13 aralkyl group or R1b and R1c together with the nitrogen atom to which they are attached denote a five- or six-membered hetercyclic ring, and X is oxygen or sulphur.
The number of nucleoside units in the oligonucleotide analogue may vary, for example from 15 to 100, according to the nature of the nucleic acid sequence to which the oligonucleotide analogue is targeted. Preferably, the oligonucleotide analogue has 15 to 40, especially 15 to 25 nucleoside units. The oligonucleotide analogue may more preferably have 15 to 20 nucleoside units for certain targets, 20 to 25 nucleoside units for other targets, 18 to 25 nucleoside units for further targets and 18 to 22 nucleoside units for yet further targets.
In an oligonucleotide analogue of the invention, the number of internucleoside linkages of formula I may vary according to the properties desired. For example, for some purposes one internucleoside linkage of formula I may suffice, while for other purposes all the internucleoside linkages may be of formula I and may be the same or different. For most purposes, up to 75%, for example up to 50%, particularly up to 25%, of the internucleoside linkages may be of formula I.
In some embodiments of the invention, at least two consecutive internucleoside linkages, for example two, three, four, five or six consecutive internucleoside linkages, which may be the same or different, in the oligonucleotide analogue are of formula I. There may be such a sequence of consecutive internucleoside linkages at each end of the oligonucleotide analogue; more usually, there is one such sequence of consecutive internucleoside linkages of formula I between sequences of nucleosides having other internucleoside linkages. In other embodiments of the invention having two or more internucleoside linkages of formula I, internucleoside linkages of formula I may alternate with other internucleoside linkages, for example along the whole length of the oligonucleotide analogue or in a region at one or both ends of the oligonucleotide analogue, or in a region in the middle of the oligonucleotide analogue.
In embodiments of the invention where not all of the internucleoside linkages are of formula I, the remaining internucleoside linkages may be natural phosphodiester linkages or other synthetic substitutes therefor such as phosphorothioate, phosphorodithioate, alkylphosphonate (xe2x80x94Oxe2x80x94P(O)(R)Oxe2x80x94), phosphoramidate, short chain alkyl, cycloalkyl, short chain heteroatomic, xe2x80x94NHCOCH2xe2x80x94, xe2x80x94CH2NHCOxe2x80x94, xe2x80x94CONHCH2xe2x80x94, xe2x80x94CH2CONHxe2x80x94, xe2x80x94CH2NHOxe2x80x94, xe2x80x94CH2N(CH3)Oxe2x80x94, xe2x80x94CH2ON(CH3)xe2x80x94, xe2x80x94CH2N(CH3)N(CH3)xe2x80x94 or xe2x80x94ON(CH3) CH2xe2x80x94 linkages, or combinations of two or more such linkages. Preferably, the remaining internucleoside linkages are phosphodiester, phosphorothioate or phosphorodithioate linkages or a mixture of two or more of these three types, particularly phosphodiester, phosphorothioate or a mixture of phosphodiester and phosphorothioate linkages. In certain especially preferred embodiments the remaining internucleoside linkages are phosphorothioate linkages.
Preferably, not more than 50% of the internucleoside linkages are phosphorothioate linkages.
In certain embodiments of the invention, the oligonucleotide comprises a region having phosphodiester and/or phosphorothioate and/or phosphorodithioate internucleoside linkages between two regions having internucleoside linkages of formula I, or a mixture thereof with phosphorothioate or phosphodiester linkages, particularly a region having phosphorothioate linkages between two regions having internucleoside linkages of formula I or a mixture thereof with phosphorothioate or phosphodiester linkages.
In some especially preferred embodiments, the oligonucleotide analogue of the invention comprises a region of at least 6 nucleosides linked by phosphorothioate linkages between two regions having nucleosides linked only by internucleoside linkages of formula I.
In oligonucleotide analogues of the invention, the nucleoside units may be natural or synthetic nucleosides having a purine or pyrimidine base such as adenine, guanine, cytosine, thymine or uracil, or an analogue of these bases such as 2-aminoadenine, 6-hydroxypurine, 5-methylcytosine, 5-propynylcytosine, 5-fluorouracil, 5-propynyluracil or dihydrouracil, attached to the Ixe2x80x2 carbon atom of a furanose sugar. As is well understood by those skilled in the art, when the oligonucleotides are for use in antisense applications, the sequence of nucleosides is chosen to be complementary to a target RNA sequence. For example, the oligonucleotide analogue of the invention may be complementary to a region of mRNA for human c-raf kinase, in which case, a preferred sequence is
5xe2x80x2-TCC CGC CTG TGA CAT GCA TT-3xe2x80x2 SEQ ID NO:1.
described as Seq. ID No. 8 in WO 95/32987 or the oligonucleotide analogue of the invention may be complementary to a region of mRNA for human PKC-xcex1, in which case a preferred sequence is
5xe2x80x2-GTT CTC GCT GGT GAG TTT CA-3xe2x80x2 SEQ ID NO:2
described as Seq. ID No. 2 in WO 95/02069.
In some oligonucleotide analogues of the invention, at least one nucleoside is modified at the 2xe2x80x2 position thereof, for example to increase binding affinity for a given target and/or to increase nuclease resistance. All of the nucleosides may be so modified, or up to 80%, for example up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, or up to 10%, of the nucleosides, may be so modified. Examples of 2xe2x80x2 modifying atoms and groups, i.e. atoms or groups which may be attached to the 2xe2x80x2 position of a nucleoside in place of a hydrogen atom or hydroxy group to effect a modification, include halogen atoms such as fluorine, chlorine and bromine atoms; C1 to C10 unsubstituted or substituted alkyl groups such as methyl, trifluoromethyl, ethyl, propyl, butyl, pentyl, hexyl, octyl or decyl; C6 to C10 aryl groups such as phenyl, tolyl or xylyl; C7 to C13 aralkyl groups such as benzyl; amino, C1 to C10 alkyl amino such as methylamino, ethylamino or octylamino; C1 to C10 alkylthio such as methylthio, ethylthio or octylthio; azide; nitrate; nitrite; cyanide; cyanate; methanesulphonate; C1 to C10 aminoalkylamino; a group of formula xe2x80x94OR2 where R2 is a C1 to C10 aliphatic group; substituted silyl; an RNA cleaving group; a cholesteryl group; a conjugate; a reporter group; an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide; and a group for improving the pharmacodynamic properties of an oligonucleotide.
Preferred modifying atoms and groups at the 2xe2x80x2 position are halogen atoms, especially fluorine, and a group of formula xe2x80x94OR2 where R2 is a C1 to C10 aliphatic group, which may be an unsubstituted or substituted C1 to C10 alkyl group such as methyl, ethyl, isopropyl, butyl, hexyl, octyl, decyl, trifluoromethyl, ethoxyethyl, methoxyethyl, or butoxyethyl, or a C2 to C6 alkenyl group such as vinyl, allyl or methallyl. Particularly preferred modifying atoms and groups are fluorine and groups of formula xe2x80x94OR2 where R2 is an unsubstituted or substituted C1 to C10 alkyl group, preferably C1 to C4 alkyl, C1 to C4 alkoxy-substituted C1 to C4 alkyl or a group of formula xe2x80x94(CH2CH2O)xe2x80x94nR3 when R3 is methyl or ethyl and n is 2 to 4. Especially preferred groups of formula xe2x80x94OR2 are those where R2 is methyl, ethyl, methoxyethyl, ethoxyethyl or a group of formula xe2x80x94(CH2CH2O)xe2x80x943CH3.
When nucleosides modified at the 2xe2x80x2 position are present, an oligonucleotide analogue of the invention may have, for example, at least two consecutive nucleosides modified at the 2xe2x80x2 position and linked by phosphodiester internucleoside linkages and/or it may have an internucleoside linkage of formula I between a nucleoside unmodified at the 2xe2x80x2 position and a 5xe2x80x2 carbon atom of a nucleoside modified at the 2xe2x80x2 position.
As is also well understood by those skilled in the art, the terminal nucleosides in the oligonucleotide analogue may have free 5xe2x80x2 and 3xe2x80x2 hydroxy groups respectively or may have either or both of these hydroxy groups replaced by a modifying group, for example a phosphate, thiol, alkylthio, thioalkyl, thiophosphate, aminoalkyl, acridinyl, cholesteryl or fluoresceinyl group.
In linkages of formula I, R1a, R1b or R1c as a substituted alkyl, alkenyl, cycloalkyl, aryl or aralkyl group may be substituted, for example, by hydroxy, C1 to C4 alkoxy, halogen (preferably chlorine or fluorine), cyano, tri(C1-C15 hydrocarbyl)silyl, or primary, secondary or tertiary amino.
In a linkage of formula I, where R1 is R1a, xe2x80x94OR1a or xe2x80x94SR1a, R1a as C1 to C10 alkyl may be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, octyl, nonyl or decyl, preferably C1 to C4 alkyl; R1a as C2 to C10 alkenyl may be vinyl, allyl, methallyl, 1-propenyl, isopropenyl, 2-butenyl, 1-butenyl, isobutenyl, pentenyl, hexenyl, octenyl or decenyl, preferably C2 to C5 alkenyl; R1a as C3 to C8 cycloalkyl may be, for example, cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, methylcyclohexyl, dimethylcyclohexyl, cycloheptyl or cyclooctyl, preferably C5 to C8 cycloalkyl; R1a as C6 to C10 aryl may be, for example, phenyl, o-tolyl, m-tolyl, p-tolyl, o-xylyl, m-xylyl, p-xylyl or naphthyl, preferably C6 to C8 aryl; R1a as C7 to C13 aralkyl may be, for example, benzyl, 4-methylbenzyl, 2-phenylethyl, 2-phenylpropyl, 3-phenylpropyl or diphenylmethyl, preferably C7 to C9 aralkyl. R1 as xe2x80x94NHR1b may be C1 to C10 alkylamino, for example, methylamino, ethylamino, isopropylamino, butylamino, pentylamino, hexylamino, octylamino or decylamino, preferably C1 to C4 alkylamino; C2 to C10 alkenylamino, for example allylamino, methallylamino, 1-propenylamino, isopropenylamino, isobutenylamino, hexenylamino, octenylamino or decenylamino, preferably C3 to C5 alkenylamino; C3 to C8 cycloalkylamino, for example, cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino, cyclooctylamino or dimethylcyclohexylamino, preferably C5 to C8 cycloalkylamino; C6 to C10 arylamino, for example phenylamino, ortho-, meta- or para-tolylamino, ortho-, meta- or para-xylylamino or naphthylamino, preferably C6 to C8 arylamino; C7 to C13 aralkylamino, for example benzylamino, 4-methylbenzylamino, 2-phenylethylamino, 3-phenylpropylamino or diphenylmethylamino, preferably C7 to C9 aralkylamino. R1 as xe2x80x94NR1bR1c may be di(C1 to C10 alkyl)amino, for example, dimethylamino, diethylamino, methylethylamino, diisopropylamino, dibutylamino or dioctylamino, preferably di(C1 to C4 alkyl) amino; N,N-di(C2-C10 alkenyl)amino, for example diallylamino, dimethallylamino, allymethallylamino, dipropenylamino, dibutenylamino, dipentenylamino, dihexenylamino, dioctenylamino or didecenylamino, preferably di(C3-C5 alkenyl)amino; N,N-di(C3-C8 cycloalkyl)amino, for example dicyclopropylamino, cyclopropylcyclopentylamino, dicyclobutylamino, dicyclopentyl amino, dicyclohexylamino, dicycloheptylamino or dicyclooctylamino, preferably N,N-di(C5-C8 cycloalkyl)amino; N-C3-C8 cycloalkyl-N-C1-C10 alkylamino, for example N-cyclopentyl-N-methylamino, N-cyclopentyl-N-ethylamino, N-cyclohexyl-N-methylamino, N-cyclohexyl-N-ethylamino, preferably N-(C5-C8 cycloalkyl)-N-C1-C4 alkylamino; N-C6-C10-aryl-N-C1-C10 alkylamino, preferably N-C6-C8-aryl-C1-C4 alkylamino, for example N-phenyl-N-methylamino, N-tolyl-N-methylamino or N-phenyl-N-ethylamino; N, N-di(C7-C13 aralkyl)amino, for example dibenzylamine, di(4-methylbenzyl)amine, di(phenylethyl)amino or di(phenylpropyl)amino, preferably N,N-di(C7-C9 aralkyl)amino; or N-C7-C13 aralkyl-N-C1-C10 alkylamino, preferably N-C7-C9 aralkyl-N-C1-C4 alkylamino, for example N-benzyl-N-methylamino or N-benzyl-N-ethylamino or a radical of a five- or six-membered N-heterocycle linked through the nitrogen atom to the indicated phosphorus atom in formula I, for example 1-pyrrolidinyl, 1-piperidyl, 1-piperazinyl or morpholino. Any of the above groups may be unsubstituted or substituted as hereinbefore described.
In certain preferred embodiments, R1 is hydrogen, hydroxy, O31 , SH, S31 , an unsubstituted or substituted C1 to C4 alkyl or phenyl group, a group of formula xe2x80x94OR1a where R1a is an unsubstituted or substituted C1 to C4 alkyl, C3 to C5 alkenyl, C5 to C8 cycloalkyl or C7 to C9 aralkyl group, or a group of formula xe2x80x94SR1a where R1a is an unsubstituted or substituted C1 to C4 alkyl or phenyl group, optional substituents being as hereinbefore described. In some especially preferred embodiments, R1 is hydrogen, hydroxy, O31 , SH, S31 , methoxy, ethoxy or 2-cyanoethoxy.
Where R1 in formula I is Oxe2x88x92, the oligonucleotide analogue of the invention may be in the form of a pharmaceutically acceptable salt, for example metal salt, preferably an alkali metal salt, or an unsubstituted or substituted ammonium salt, for example a mono-, di- or tri-C1 to C10 alkyl- or hydroxyalkyl-ammonium salt, a N-ethylpiperidinylium salt or a N,N1-dimethylpiperazinylium salt. In especially preferred embodiments where R1 is Oxe2x88x92, the oligonucleotide analogue is in the form of the sodium or ammonium salt.
Where the phosphorus atom in formula I is a chiral centre, differences may be observed in hybridisation and nuclease resistance properties and in biological efficacy depending on the stereochemistry at phosphorus.
An oligonucleotide analogue of the invention may be represented by the formula Vxe2x80x94L"Parenopenst"Vxe2x80x94L"Parenclosest"nV where n is a number from 8 to 198, each V is independently a residue of a natural or synthetic nucleoside, each of the n+2 residues V being the same as, or different from, an adjacent residue V, and each L is an internucleoside linkage, each of the n+1 linkages L being the same as, or different from, an adjacent linkage L, at least one L being of formula I.
The present invention also provides a method of preparing an oligonucleotide analogue having at least one internucleoside linkage of formula I, for example an oligonucleotide having 2 to 200 nucleoside units, such as an oligonucleotide analogue as hereinbefore described, which comprises (i) carrying out a coupling reaction or successive coupling reactions between (A) a natural or synthetic nucleoside or oligonucleotide having a 5xe2x80x2-hydroxyl group and (B) a natural or synthetic nucleoside or dinucleotide having at the 3xe2x80x2-position thereof a group reactive with said 5xe2x80x2-hydroxyl group until an oligonucleotide having the desired number of nucleosides is obtained, in at least one of said coupling reactions (B) being a nucleoside of formula 
where B1 is a nucleoside base radical, R4 is a hydroxy-protecting group, R5 is hydrogen, hydroxy or a 2xe2x80x2 modifying atom or group, M+ is a metal or unsubstituted or substituted ammonium ion or a cation of a heterocyclic base such as pyrrolidine, piperidine, N-ethylpiperidine, N,N1-dimethylpiperazine, morpholine or 1,8 diazabicyclo[5.4.0]undec-7-ene (DBU) and X is oxygen or sulphur, and being reacted with (A) in the presence of a sterically hindered organic acid halide or anhydride to form an oligonucleotide analogue having a phosphinate internucleoside linkage of formula 
where X is oxygen or sulphur, and (ii)(a) oxidising the phosphinate linkage or (b) sulphurising the phosphinate linkage, or (c) reacting the phosphinate linkage with a compound of formula R1aY where R1a is as hereinbefore defined and Y is a leaving atom or group or (d) oxidising and reacting the phosphinate linkage with an alcohol of formula R1aOH or an amine of formula R1bNH2 or R1bR1c NH where R1a, R1b and R1c are as hereinbefore defined, or (e) silylating the phosphinate linkage and reacting the silylated linkage with a thioalkylating or thioarylating agent to give a phosphinate linkage of formula I where R1 is xe2x80x94SR1a where R1a is as hereinbefore defined.
The hereinbefore defined method may be carried out in solution or on a solid carrier, for example using known procedures for oligonucleotide synthesis. The oligonucleotide analogue obtained by this method may be further reacted to replace the protecting group R4 by hydrogen or, where R4 is on a terminal nucleoside in the oligonucleotide analogue, by a 5xe2x80x2 modifying group as hereinbefore described.
Oligonucleotide analogues of the invention may be prepared by solid phase synthesis, for example using H-phosphonate, phosphotriester or phosphoramidite methods, or a mixture of two or more thereof, for example automatically using commercially available nucleic acid synthesisers. A solid phase synthetic method may comprise carrying out successive coupling reactions (i) as hereinbefore described and step (ii) as hereinbefore described with the nucleoside or oligonucleotide (A) attached to the solid support, then (iii) detaching the oligonucleotide from the solid support and removing protecting groups to give an oligonucleotide having a terminal 5xe2x80x2 free hydroxyl group and (iv) optionally reacting the 5xe2x80x2 free hydroxyl group to introduce a modifying group at the terminal 5xe2x80x2 position. In the nucleoside of formula II, B1 may be a purine or pyrimidine base or analogue thereof as hereinbefore described. Compounds where B1 is a natural nucleoside base, more preferably pyrimidine base, especially thymine, are preferred. R4 may be any hydroxy-protecting group capable of protecting the 5xe2x80x2 hydroxyl group against undesired reaction. Such groups are well known and include C1 to C10 aliphatic, e.g. alkyl, groups; C3 to C8 cycloaliphatic, e.g. cycloalkyl, groups; C6 to C10 aromatic, e.g. aryl, groups; C7 to C40 araliphatic, e.g. aralkyl or C1 to C4 alkoxy-substituted aralkyl, groups; groups of formula xe2x80x94COR6 or xe2x80x94SO2R6 where R6 is a C1 to C10 aliphatic group, a C3 to C8 cycloaliphatic group, a C6 to C10 aromatic group or a C7 to C40 araliphatic group; and tri(C1-C15 hydrocarbyl)silyl groups. Preferably R4 is a 5xe2x80x2 protecting group conventionally used in oligonucleotide synthesis, especially a methoxytrityl, dimethoxytrityl or tris tert-butyltrityl group. R5 as a 2xe2x80x2 modifying atom or group may be such an atom or group as hereinbefore described; preferably R5 is hydrogen. M+ may be, for example, an alkali metal ion or, preferably, an unsubstituted ammonium ion, a mono-di- or tri-C1 to C10 alkyl- or hydroxyalkyl-ammonium ion or a cation of a heterocyclic base such as pyrrolidine, piperidine, N-ethylpiperidine, N,N-dimethylpiperazine, morpholine or DBU. An especially preferred M+ is a triethylammonium ion.
Preferred stereoisomers of nucleosides of formula II are those of formula 
where B1, R4, R5, X and M+ are as hereinbefore defined.
Nucleosides of formula II may be prepared by a) reacting a compound of formula 
where B1 and R4 are as hereinbefore defined, R5a is hydrogen, fluorine or xe2x80x94OR2 where R2 is as hereinbefore defined and L is a leaving atom or group, preferably an iodine atom, with ethyl (1,1 -diethoxyethyl)phosphinate in the presence of a base such as potassium bis(trimethylsilyl)amide in tetrahydrofuran (THF) at xe2x88x9280xc2x0 C. to 40xc2x0 C. to give a compound of formula 
where B1 and R5a are as hereinbefore defined, b) reacting the compound of formula IV with trimethylsilyl chloride in chloroform containing 1% ethanol under argon at ambient temperature to replace the protecting ketal group attached to phosphorus by hydrogen, c) reacting the product from b) with acetone in the presence of titanium (IV) isopropoxide in dry THF at ambient temperature to give a compound of formula 
d) reacting the compound of formula V with tetra-n-butylammonium fluoride and acetic acid in THF at ambient temperature to remove the tert-butyldiphenylsilyl protecting group, e) reacting the resulting 5xe2x80x2-hydroxy-containing compound with a compound of formula R4Y, where R4 is as hereinbefore defined and Y is halogen, in the presence of an organic base to give a compound of formula 
where B1, R4 and R5a are as hereinbefore defined, f) reacting the compound of formula VI with an alkali metal methoxide in anhydrous methanol, or with DBU in water, at ambient temperature to remove the ethyl group and replace the xe2x80x94C(CH3)2 OH group by hydrogen, g) if desired, treating the product with ammonia or an amine to form the corresponding unsubstituted or substituted ammonium salt, and h) if desired, sulphurising the product to give a nucleoside of formula II in which X is sulphur, for example by reaction with pivaloylchloride followed by (CH3)3 Sixe2x80x94Sxe2x80x94Si(CH3)3 using the procedure described in J. Org. Chem. 1995, 60, 8241.
The compounds of formulae IV, V or VI may be reacted to introduce, or introduce a different, modifying atom or group R5 at the 2xe2x80x2 position using, for example, a conventional procedure for introducing such a 2xe2x80x2 modifying atom or group into a nucleoside.
Compounds of formula III may be prepared by reducing an aldehyde of formula 
where B1 and R5a are as hereinbefore defined, prepared according to the method described in WO 92/20823, to the corresponding alcohol by reaction with NaBH4 in anhydrous ethanol at ambient temperature and reacting the alcohol with methyltriphenoxyphosphonium iodide in the presence of 2,6-lutidine in dry dimethyl formamide at 0xc2x0 C. to 30xc2x0 C.
Ethyl(1,1-diethoxyethyl)phosphinate may be prepared as described in EP 0 307 362.
In a typical procedure using solid phase synthesis, a natural or synthetic nucleoside having a protected 5xe2x80x2 hydroxyl group is covalently linked at the 3xe2x80x2 position to an inert silica-based support, such as controlled pore glass (CPG), containing long chain alkylamino groups, using a linker such as succinic anhydride to give a 3xe2x80x2-terminal nucleoside attached to the solid support. The solid support may also contain groups to act as 3xe2x80x2 terminal modifying groups for the desired oligonucleotide. The protecting group, for example a dimethoxytrityl group, on the 5xe2x80x2 hydroxy of the attached terminal nucleoside is then removed to give a free 5xe2x80x2 hydroxy group. The terminal nucleoside is then coupled with a natural or synthetic nucleoside or dinucleotide (B) having a protected, e.g. dimethoxytrityl-protected, 5xe2x80x2 hydroxyl group and, at the 3xe2x80x2 position a group which is reactive with, or activatable to be reactive with, the 5xe2x80x2 free hydroxyl group on the terminal nucleoside to give a dimeric oligonucleotide (or, where (B) is a dinucleotide, a trimeric oligonucleotide) attached to the solid support. After the 5xe2x80x2 protecting group on the attached oligonucleotide has been removed, the reaction cycle with a natural or synthetic 5xe2x80x2 protected nucleoside or dinucleotide (B) having a 3xe2x80x2 reactive group is repeated until an oligonucleotide having the desired number of nucleosides has been synthesised.
For the formation of internucleoside linkages other than those of formula I, the reactive group, or the group activatable to be reactive, at the 3xe2x80x2 position of the nucleoside or dinucleotide (B) is chosen in accordance with conventional oligonucleotide synthesis procedures and may be, for example, an H-phosphonate group, a phosphoramidite group or a phosphodiester group. The coupling reactions and, where necessary, subsequent oxidation, sulphurisation or other treatment, to form these internucleoside linkages, for example phosphotriester, phosphorothioate or phosphorodithioate linkages, may be carried out using conventional procedures.
In preparing an oligonucleotide analogue of the invention, at least one of the coupling reactions is carried out using as (B) a nucleoside of formula II, which is reacted with the nucleoside or oligonucleotide (A), which in solid phase synthesis is attached to the solid support, in the presence of a sterically hindered organic acid halide or anhydride, for example pivaloyl chloride, adamantoyl chloride, 2,4,6-triisopropylbenzenesulphonyl chloride, diphenylphosphinic chloride, bis(2-oxo-3-oxazolidinyl) phosphinic chloride, 2-chloro-2-oxo-5,5-dimethyl-1,3,2-dioxaphosphinane or bis(pentafluorophenyl) anhydride. Preferably, the reaction is carried out in the presence of a heterocyclic base having a tertiary nitrogen atom in the ring or an oxide of such a base, for example, pyridine, quinoline, N-methylimidazole or pyridine-N-oxide and, especially, an organic solvent such as acetonitrile. The coupling reaction may be carried out at ambient or moderately elevated temperatures, for example up to 50xc2x0 C.
The phosphinate internucleoside linkage of formula IA formed by a coupling reaction using as (B) a nucleoside of formula II may be oxidised or sulphurised before the next coupling reaction is carried out or, preferably, after an oligonucleotide analogue with the desired number of nucleosides has been synthesised, when it may be oxidised or sulphurised together with one or more other phosphinate internucleoside linkages formed by other coupling reactions using a nucleoside of formula II or phosphite linkages formed by coupling reactions using a 3xe2x80x2 H phosphonate-substituted nucleoside. Oxidation (ii)(a) may be effected by treatment with iodine and water, or with tert-butyl hydroperoxide, for example using conventional procedures for oxidation of phosphite internucleoside linkages. Sulphurisation (ii)(b) may be effected by treatment with sulphur in the presence of a tertiary amine in an organic solvent, usually carbon disulphide, for example using known procedures.
The reaction (ii)(c) of the phosphinate internucleoside linkage of formula IA with a compound of formula R1aY where R1a and Y are as hereinbefore defined, Y preferably being a halogen atom or a trifluoromethanesulphonate group, may be carried out, where R1a is alkyl, cycloalkyl or aralkyl, using known alkylation procedures, for example by reacting the phosphinate linkage of formula IA with R1aY where Y is halogen in the presence of a strong base such as sodium hydride. Where R1aY is an alkenyl or aryl halide or triflate, the reaction between the phosphinate linkage and R1aY may be carried out using known procedures, for example in the presence of a palladium catalyst such as Pd(PPh3)4 and a tertiary amine, for example as described by Y.Xu et al, Tetrahedron Lett., 30, 949(1989) or K. S. Petrakis et al, J. Am. Chem. Soc., 109, 2831 (1987).
The oxidative reaction (ii)(d) of the phosphinate internucleoside linkage of formula IA with an alcohol of formula R1aOH, before the next coupling reaction or after an oligonucleotide with the desired number of nucleosides has been synthesised, may be carried out by reaction with an oxidant such as iodine, carbon tetrachloride or bromotrichloromethane in the presence of the alcohol R1aOH and a base such as pyridine, for example under conventional conditions for oxidation of phosphite internucleoside linkages.
The oxidative reaction (ii)(d) of the phosphinate internucleoside linkage of formula IA may be effected, before the next coupling reaction or after an oligonucleotide with the desired number of nucleosides has been synthesised, with an amine of formula R1bNH2 or R1bR1cNH where R1b and R1c are as hereinbefore defined, and carbon tetrachloride or bromotrichloromethane or iodine to give an oligonucleotide analogue of the invention in which R1 is xe2x80x94NHR1b or xe2x80x94NR1bR1c respectively. The reaction may be carried out using known conditions and procedures for Atherton-Todd reactions.
The reaction (ii)(e) of the phosphinate linkage of formula IA may be carried out by silylating the linkage using known silylation procedures, for example using a trialkylsilyl halide and a base such as triethylamine, and reacting the silylated linkage with a thioalkylating or thioarylating agent such as a thiosulphonate of formula ArSO2SR1a where R1a is as hereinbefore defined and Ar is an aromatic group such as phenyl or tolyl. Suitable procedures for this reaction are described by W. K. D. Brill, Tetrahedron Lett, 36, 703 (1995).
It will be apparent to those skilled in the art that in reacting the phosphinate linkage of formula IA to replace the hydrogen atom attached to phosphorus by a group R1 having a reactive substituent such as amino, the substituent should be protected during the reaction to introduce R1 if it is reactive under the conditions of that reaction and subsequently deprotected.
When an oligonucleotide analogue having the desired number of nucleosides has been synthesised on a solid support, it is detached from the solid support, for example using conventional methods such as treatment with concentrated aqueous ammonia, which treatment also removes a protecting group which may have been present on an exocyclic nitrogen atom in one or more of the nucleosides used in the synthesis of the oligonucleotide, before or after treatment to remove hydroxy-protecting groups such as dimethoxytrityl groups, which may also be carried out using conventional methods, for example by treatment with an aqueous organic acid such as trifluoroacetic acid.
Before or after detachment of the oligonucleotide from the solid support, the terminal 5xe2x80x2 hydroxyl generated on deprotection can be reacted to introduce a 5xe2x80x2 terminal modifying group, such as a phosphate group or other 5xe2x80x2 modifying group as hereinbefore described, for example using the procedures described by Beaucage and lyer, Tetrahedron 49, 1925-63 (1993).
In a modification of the synthetic method hereinbefore described, the nucleoside of formula II may be replaced by a dinucleotide of formula 
where B1, R1, R4 and R5 are as hereinbefore defined, B2 is a nucleoside base radical, which may be a radical of a natural or synthetic nucleoside base as hereinbefore described for B1, R6 is hydrogen, hydroxy or a 2xe2x80x2 modifying atom or group as hereinbefore defined for R5, and R7 is a group reactive with, or activatable to be reactive with, a 5xe2x80x2 hydroxyl group in a nucleoside.
In this modification, the group R7O in the dinucleotide of formula VIII may be a H-phosphonate group, in which case the dinucleotide of formula VII may be reacted with the nucleoside or oligonucleotide (A), which in solid phase synthesis is attached to the solid support, in the presence of a sterically hindered organic acid halide, for example using conventional procedures for oligonucleotide synthesis using 3xe2x80x2 H-phosphonates, to form a phosphite internucleoside linkage which may then be oxidised, sulphurised or reacted with a compound of formula R1aY or subjected to another of the reactions (ii)(a) to (ii)(e) as hereinbefore described for the phosphinate internucleoside linkage of formula IA formed by reaction of the nucleoside of formula II with (A).
In this modification, the group R7O in the dinucleotide of formula VIII may alternatively be a phosphoramidite group, in which case the dinucleotide of formula VIII may be reacted with the nucleoside or oligonucleotide (A), for example using conventional procedures for oligonucleotide synthesis using 3xe2x80x2 phosphoramidites.
In another alternative embodiment of this modification, the group R7O in the dinucleotide of formula VIII may be a phosphodiester group, in which case the dinucleotide of formula VIII may be reacted with the nucleoside or oligonucleotide (A), for example using conventional procedures for oligonucleotide synthesis using 3xe2x80x2 phosphodiesters.
Dinucleotides of formula VIII may be prepared by reacting a nucleoside of formula II with a nucleoside of formula 
where B2 and R6 are as hereinbefore defined and R8 is a hydroxy-protecting group, in the presence of a dehydrating coupling reagent e.g. a carbodiimide or a sterically hindered organic acid halide or anhydride, to give a dinucleotide of formula 
and, optionally after subjecting the internucleoside linkage in formula X to any of reactions (ii)(a) to (ii)(e) as hereinbefore described, converting the R8Oxe2x80x94 group into a R7Oxe2x80x94 group.
The hydroxy-protecting group R8 may be chosen from groups hereinbefore specified for R4. Preferably R8 is a 3xe2x80x2 protecting group conventionally used in nucleoside chemistry, especially a tert-butyldiphenylsilyl group.
Nucleosides of formula IX are 3xe2x80x2 protected natural or synthetic nucleosides which may have hydrogen, hydroxy or a 2xe2x80x2 modifying atom or group at the 2xe2x80x2 position. Such nucleosides are known or may be prepared by known methods.
The reaction between the nucleoside of formula II and the nucleoside of formula IX in the presence of a sterically hindered organic acid halide is preferably carried out in the presence of a heterocyclic base or oxide thereof and an organic solvent as hereinbefore described for the reaction of the nucleoside of formula II with the nucleoside or oligonucleotide (A).
The conversion of the group R8Oxe2x80x94 into a group R7Oxe2x80x94 where R8 and R7 are as hereinbefore defined may be carried out using conventional methods for converting a protected 3xe2x80x2 hydroxyl group into a group reactive with, or activatable to be reactive with, a 5xe2x80x2 hydroxyl group, such as a H-phosphonate, phosphoramidite or phosphodiester group. For example, the protecting group R8 may be removed to generate a free 3xe2x80x2 hydroxyl, which may then be reacted with an aliphatic bis(N,N-dialkyl)phosphoramidite such as 2-cyanoethyl bis(N,N-diisopropyl)phosphordiamidite to form a 3xe2x80x2 phosphoramidite group.
Dinucleotides of formulae VIII where one or each of R5 and R6 is 2xe2x80x2 modifying atom or group as hereinbefore defined, particularly a group of formula xe2x80x94OR2 as hereinbefore defined, are novel. Dinucleotides of formula X are novel. Thus the invention also provides novel dinucleotides of formula 
where B1, B2, R1, R4 are as hereinbefore defined, R5 and R6 are as hereinbefore defined except that where R1 is other than hydrogen at least one of R5 and R6 is a 2xe2x80x2 modifying atom or group as hereinbefore defined, and R9 is R7 or R8 as hereinbefore defined, especially those where R5 is hydrogen or hydroxy and R6 is a 2xe2x80x2 modifying atom or group as hereinbefore defined, particularly a group of formula xe2x80x94OR2 as hereinbefore defined.
Dinucleotides of formula VIII where R1 is C1 to C10 alkoxy may also be prepared by reacting a nucleoside of formula 
where B1, R4 and R5 are as hereinbefore defined, with a nucleoside of formula IX in the presence of a tertiary amine such as dimethylaminopyridine and a dehydrating agent such as dicyclohexylcarbodiimide (DCC), to give a dinucleotide of formula X, which is then treated as hereinbefore described to give a dinucleotide of formula VIII. The reaction between the nucleosides of formulae XII and IX may be carried out in a solvent such as THF at ambient temperature.
Nucleosides of formula XII can be prepared by treating nucleosides of formula II (salt forms of acids of formula XII) with acid using conventional procedures.
Oligonucleotides having at least one internucleoside linkage of formula I, for example an oligonucleotide having 2 to 200 nucleoside units, such as an oligonucleotide analogue as hereinbefore described, may also be prepared by subjecting a nucleoside having a protected 5xe2x80x2 hydroxy group and, at the 3xe2x80x2 position, a group of formula 
where R1a is as hereinbefore defined, R10 and R11 are each independently an unsubstituted or substituted C1 to C10 alkyl, C2 to C10 alkenyl, C4 to C10 cycloalkylalkyl, C6 to C10 aryl or C7 to C13 aralkyl group, or R10 is said group and R11 is hydrogen, or R10 and R11 together with the nitrogen atom to which they are attached denote a five- to thirteen-membered heterocyclic ring, to a nucleoside coupling reaction with a natural or synthetic nucleoside or oligonucleotide having a free 5xe2x80x2 hydroxy group, to form an oligonucleotide precursor having an internucleoside linkage of formula 
where R1a is as hereinbefore defined, and converting the precursor into an oligonucleotide having an internucleoside linkage of formula 
where R1a is as hereinbefore defined and X is oxygen or sulphur by oxidising the precursor to give an oligonucleotide having an internucleoside linkage of formula XV where X is oxygen or sulphurising the precursor to give an oligonucleotide having an internucleoside linkage of formula XV where X is sulphur.
The reaction to form the precursor having a linkage of formula XIV may be carried out in the presence of an amine-protonating coupling catalyst (activating agent) such as tetrazole or 5-(4-nitrophenyl)tetrazole. The reaction may be carried out at xe2x88x9220 to 50xc2x0 C., preferably at room temperature. The oxidation or sulphurisation of the resulting precursor may be effected by methods used for oxidation or sulphurisation respectively of phosphite internucleoside linkages. Thus oxidation may be effected by treatment with iodine and water, or with a hydroperoxide such as tert-butyl hydroperoxide, for example using conditions and procedures known for oxidation of phosphite internucleoside linkages in oligonucleotide synthesis. Sulphurisation may be effected by treatment with sulphur in the presence of a tertiary amine in an organic solvent, usually carbon disulphide, by treatment with [3H] 1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent) or by treatment with tetraethylthiuram, for example using procedures known for sulphurisation of phosphite internucleoside linkages.
Nucleosides having a protected 5xe2x80x2 hydroxy group and, at the 3xe2x80x2 position, a group of formula XIII, may be prepared by reacting a nucleoside having a protected 5xe2x80x2 hydroxy group and, at the 3xe2x80x2 position, a group of formula 
where R1a is as hereinbefore defined and Z is halogen, with a compound of formula 
where R10 and R11 are as hereinbefore defined. The reaction may be carried out in an organic solvent, for example a halogenated hydrocarbon such as chloroform, in the presence of a tertiary nitrogen base such as pyridine, and at a temperature from xe2x88x9278xc2x0 C. to 50xc2x0 C., preferably from xe2x88x9230xc2x0 C. to 25xc2x0 C.
Nucleosides having a protected 5xe2x80x2 hydroxy group and, at the 3xe2x80x2 position, a group of formula XVI may be prepared by non-oxidative halogenation of a nucleoside having a protected 5xe2x80x2 hydroxy group and, at the 3xe2x80x2 position, a group of formula 
The non-oxidative halogenation may be carried out by reaction with a non-oxidative halogenating agent, for example a halophosphorane such as triphenyldichlorophosphorane or dichlorotris(2,4,6-tribromophenoxy)phosphorane in the presence of a base, preferably a tertiary nitrogen base such as pyridine, in an organic solvent, which may be pyridine but is preferably a halohydrocarbon such as chloroform, at a temperature from xe2x88x9220xc2x0 C. to 60xc2x0 C., preferably from 0xc2x0 C. to 50xc2x0 C.
Nucleosides having a protected 5xe2x80x2 hydroxy group, and at the 3xe2x80x2 position, a group of formula XVIII may be prepared as described in WO 96/08503.
When the oligonucleotide having a linkage of formula XV is formed on a solid support, it may be treated to remove the 5xe2x80x2 protecting group and the resulting 5xe2x80x2 hydroxy-terminated oligonucleotide subjected to successive coupling cycles with a natural or synthetic nucleoside or oligonucleotide having a protected 5xe2x80x2 hydroxyl group and, at the 3xe2x80x2 position, a group reactive with, or activatable to be reactive with, the free 5xe2x80x2 hydroxy group on the deprotected oligonucleotide attached to the solid support, until an oligonucleotide of the desired length is obtained. Thus the oligonucleotide having a linkage of formula XV may be coupled with a nucleoside or oligonucleotide having a 3xe2x80x2 phosphoramidite, H-phosphonate, phosphodiester group or 3xe2x80x2 group of formula XIII and a protected 5xe2x80x2 hydroxyl group, to give a chain-extended oligonucleotide which may in turn be further chain extended by further such alternative reactions until an oligonucleotide of the desired length is obtained. Where a nucleoside or oligonucleotide having a 3xe2x80x2 phosphoramidite, H-phosphonate or phosphodiester group is used, the coupling reaction may be carried out using procedures known in oligonucleotide synthesis. Where a nucleoside having a 3xe2x80x2 group of formula XIII is used, the coupling reaction may be carried out as hereinbefore described. Thus, where a 3xe2x80x2 phosphoramidite or a 3xe2x80x2 group of formula XIII is used, a coupling cycle involves an oxidation or sulphurisation while where a 3xe2x80x2H-phosphonate is used, oxidation or sulphurisation is effected after chain extension is complete, and where a 3xe2x80x2 phosphodiester is used no oxidation is required.
The oligonucleotide analogues of the invention can be used in therapeutics, for example in the treatment of a human or other animal suffering from a disease which is modulated by a protein, or in the treatment of viruses such as influenza, herpes and HIV. Accordingly, the present invention also provides a pharmaceutical composition comprising as active ingredient an oligonucleotide analogue of the invention. Optimum dosages and treatment schedules can readily be determined by those skilled in the art. When administered to mammals of about 70 kg weight, the dose can be, for example, 0.01 to 1000 mg per day. It will generally be preferred to administer therapeutic agents in accordance with the invention internally, for example orally, by inhalation, intravenously or intramuscularly. Other methods of administration, such as transdermal, topical or inter-lesional methods, and by inclusion in suppositries, can also be useful. Use in conduction with pharmacologically acceptable carriers is preferred for some therapeutic treatments.
The oligonucleotide analogues according to the invention have a surprisingly high stability to degradation by nucleases. A very good pairing with complementary nucleic acid strands, particularly of the RNA type, is also observed. The oligonucleotide analogues according to the invention are therefore particularly suitable for antisense technology, i.e. for inhibition of the expression of undesired protein products due to the binding to suitable complementary nucleotide sequence in nucleic acids (see EP 0 266 099, WO 87/07300 and WO 89/08146). They can be employed for the treatment of infections and diseases, for example by blocking the expression of bioactive proteins at the nucleic acid stage (for example oncogenes). The oligonucleotide analogues according to the invention are also suitable as diagnostics and can be used as gene probes for the detection of viral infections or of genetically related diseases by selective interaction at the single or double-stranded nucleic acid stage. In particularxe2x80x94due to the increased stability to nucleasesxe2x80x94diagnostic use is not only possible in vitro but also in vivo (for example tissue samples, blood plasma and blood serum). Use possibilities of this type are described, for example, in WO 91/06556.
The novel dinucleotides of formula XI can be used as pharmaceuticals, for example as antiviral agents.
The pharmacologically active oligonucleotide analogues and dinucleotides according to the invention can be used in the form of parenterally administrable preparations or of infusion solutions. Solutions of this type are preferably isotonic aqueous solutions or suspensions, it being possible to prepare these before use, for example in the case of lyophilised preparations which contain the active substance on its own or together with a carrier, for example mannitol. The pharmaceutical preparations can be sterilised and/or contain excipients, for example preservatives, stabilisers, wetting and/or emulsifying agents, solubilisers, salts for regulating the osmotic pressure and/or buffers. The pharmaceutical preparations, which if desired can contain further pharmacologically active substances such as, for example, antibiotics, are prepared in a manner known per se, for example by means of conventional dissolving or lyophilising processes, and contain about 0.1% to 90%, in particular from about 0.5% to about 30%, for example 1% to 5% of active substance(s).
This invention is illustrated by the following Examples.
Compounds used in the Examples, and precursors thereof, are prepared as follows. All 31P data for these compounds and those of the Examples are for 1H decoupled. 
To a solution of an aldehyde of formula XIII where R2 is hydrogen, B1 is I-thyminyl and R1 is tert-butyl diphenylsilyl, prepared as described in WO 92/20823, (11.2 g 23 mmol) in anhydrous ethanol (120 ml) at room temperature is added NaBH4 (865 mg, 23 mmol) portionwise over 5 minutes. After 1 hour, the reaction mixture is quenched with water, diluted with ethylacetate (500 ml) and washed with water (2xc3x9750 ml). After back extraction of the aqueous phase, the combined organic phase is dried (MgSO4) and concentrated to give Compound A as a white solid.
1H nmr (CDCl3, 400 MHz) xcex49.10 (1H, s, NH) 7.65 (4H, d, Ar 4xc3x97CH ortho), 7.40 (7H, m, Ar 4xc3x97CH meta, 2xc3x97CH para+H6) 6.13 (1H, t, H1xe2x80x2) 4.00 (1H, dd, H5xe2x80x2), 3.93 (1H, m, H4xe2x80x2) 3.82 (1H, dd, H5xe2x80x2), 3.62 (2H, m, CH2OH) 2.60 (1H, m, H3xe2x80x2), 2.32 (1H, m, H2xe2x80x2), 2.12 (1H, m, H2xe2x80x2) 1.62 (3H, s, T-CH3) and 1.10 (9H, s, tBu) ppm. 
To a solution of Compound A (9 g, 18.1 mmol) in dry DMF (100 ml) at 0-5xc2x0 C. is added 2,6-lutidine (4.25 ml, 36.5 mmol) followed by methyltriphenoxyphosphonium iodide (9.45 g, 20.9 mmol). The resulting mixture is allowed to warm to room temperature. After 1 hour the mixture is diluted (200 ml ethyl acetate) and washed with 0.1N NaS2O3 (2xc3x9720 ml), 0.5N Hydrochloric acid (2xc3x9720 ml) and water (2xc3x9720 ml). Drying, concentration and purification by flash silica column chromatography (gradient elution chloroform:ethylacetate 20:1-7:1) gives Compound B as a white solid.
1H nmr (CDCl3, 400 MHz) xcex410.2 (1H, s, NH) 7.66 (4H, d, 4xc3x97CH ortho), 7.40 (7H, M, 4xc3x97CH meta, 2xc3x97CH para+H6) 6.19 (1H, t, H1xe2x80x2) 4.02 (1H, dd, H5xe2x80x2), 3.82 (1H, m, H4xe2x80x2) 3.78 (1H, dd, HSxe2x80x2), 3.17 (1H, dd, CH2I) 3.10 (1H, dd CH2I), 2.68 (1H, m, H3xe2x80x2), 2.30 (1H, m, H2xe2x80x2), 2.23 (1H, m, H2xe2x80x2) 1.66 (3H, s, CH3-T), 1.10 (9H, s, tBu) ppm. 
To a solution of ethyl (1,1-diethoxyethyl)phosphinate (5.51 g, 26.2 mmol) in dry THF (170 ml), under argon, at xe2x88x9278xc2x0 C. is added a solution of potassium bis(trimethylsilyl)amide (34.6 ml, 0.75M solution in toluene) dropwise over 5 minutes. The resulting solution is stirred at xe2x88x9278xc2x0 C. for 1 hour. A solution of Compound B (5.0 g, 8.25 mmol) in dry THF (20 ml) is then added dropwise over 5 minutes. Stirring is continued at xe2x88x9278xc2x0 C. for 1 hour before warming to room temperature over 2 hours. Saturated aqueous ammonium chloride (50 ml) is then added and the whole mixture extracted with ethyl acetate (500 ml). The organic phase is washed with saturated ammonium chloride (2xc3x9750 ml) and water (2xc3x9750 ml), dried over magnesium sulphate and concentrated. Purification by flash silica column chromatography (eluant ethylacetate:ethanol 30:1) gives Compound C as a 1:1 mixture of diastereoisomers epimeric at phosphorous. 
Trimethylsilylchloride (4.44 ml, 35 mmol) is added dropwise (2 minutes) at room temperature to a stirred solution of Compound C (2.4 g, 3.5 mmol) in chloroform (25 ml) containing ethanol (1%) under argon. After standing at xe2x88x9220xc2x0 C. for 60 hours, a further portion of trimethylsilylchloride (2.22 ml, 17.5 mmol) is added along with ethanol (200 xcexcl) and the resulting solution stirred at room temperature for 7 hours. Concentration and co-evaporation with chloroform (50 ml) gives a white solid which is purified by flash silica column chromatography (eluant chloroform: ethanol 13:1) to give Compound D as a white solid isolated as a 1:1 mixture of diastereoisomers. 
To a solution of Compound D (1.2 g, 2.1 mmol) in dry THF (30 ml) containing acetone (3.2 ml) is added in titanium (IV) isopropoxide (738 xcexcl, 2.48 mmol). After 15 minutes, concentration and passage through a short column of silica (eluant ethyl acetate:ethanol 4:1) (500 ml) gives Compound E isolated as a mixture of 2 diastereoisomers.
31P nmr 1H decoupled (CDCl3, 162 MHz) xcex455.0, 54.7 ppm.
Found: C, 57.7; H, 7.05; N, 4.05% C32H45N2O7PSi.2H2O requires C, 57.8; H, 7.4; N, 4.2% 
To a solution of Compound E (1.02 g, 1.62 mmol) and acetic acid (92 xcexcl, 16.1 mmol) in THF (10 ml) is added a solution of tetra-n-butyl ammonium fluoride (1.63 ml, 1.0 Molar). After stirring at room temperature for 1 hour, the mixture is concentrated and co-evaporated with chloroform (50 ml). Purification by flash silica column chromatography (eluant chloroform:ethanol 9:1) gives Compound F isolated as a mixture of two diastereoisomers.
Found: C, 45.55; H, 6.85; N, 6.4% C16H27N2O7P.1 2/3H2O requires C, 45.7; H, 7.25; N, 6.6% 31P nmr 1H decoupled (CDCl3, 162 MHz) xcex456.7, 56.5 ppm. 
To a solution of Compound F (550 mg, 1.41 mmol) in pyridine (10 ml) is added dimethoxytritychloride (958 mg, 2.83 mmol). After stirring at room temperature for 20 hours, concentration and purification by flash silica column chromatography (eluant chloroform, methanol, triethylamine 100:5:1) gives Compound G, isolated as a mixture of 2 diastereoisomers.
31P nmr 1decoupled (CDCl3, 162 MHz) xcex454.9, 54.7 ppm. 
To a solution of Compound G (0.85 g, 1.22 mmol) in anhydrous methanol (10 ml) is added sodium methoxide (1.5 ml 4.4N solution in methanol). After stirring for 16 hours at room temperature, concentration and purification by flash silica column chromatography (gradient elutionxe2x80x94chloroform, methanol, triethylamine 100:20:1-100:35:1), followed by further purification by passing a solution of the product in aqueous 0.5% triethylamine through a Dowex 50W-X2 ion exchange column (triethylamine form) gives, after concentration, Compound H.
31P nmr 1H decoupled (CD3OD, 162 MHz) xcex423.7 ppm. 
Compound J is prepared as described in Example 98 of WO 96/08503.
In the formulae of Compounds K to M, T is 1-thyminyl, and DMTr is dimethoxytrityl. 
To a solution of Compound H (500 mg, 0.71 mmol) and dicyclohexylcarbodiimide (189 mg, 0.92 mmol) in dry THF (5.4 ml) under argon at room temperature is added 3-hydroxy propionitrile (58 xcexcl, 0.85 mmol). The resulting solution is heated at 55xc2x0 C. for 2 hours. After cooling, the mixture is filtered and diluted with ethyl acetate (20 ml) and washed with water and brine, dried over Na2SO4, filtered and concentrated. The resulting product is taken up in dichloromethane (5 ml) and filtered and concentrated, this process being repeated as required to remove dicyclohexyl urea, to give Compound K, isolated as a mixture of diastereoisomers at phosphorus.
31P nmr (1H decoupled) (CDCl3, 162 MHz) xcex437.4, 37.3 ppm. 
To a solution of carefully dried Compound K (71 mg, 220 xcexcmol) in deuterochloroform (0.5 ml) containing pyridine (80 xcexcl, 1 mmol) is added dichlorotriphenylphosphorane (113 mg, 350 xcexcmol). The resulting mixture is shaken to dissolve the phosphorane and then allowed to stand at ambient temperature. The progress of the reaction is monitored by 31P nmr. The product is Compound L. After 16 hours, additional dichlorotriphenylphosphorane (28 mg, 87 xcexcmol) is added. After an additional 24 hours, 31P nmr shows the reaction to be 95% complete. A total 56 xcexcl (0.67 mmol) of pyrrolidine is added in portions to the crude reaction mixture at xe2x88x9230xc2x0 C. The resulting mixture is allowed to warm to room temperature and then diluted with dichloromethane (20 ml), washed twice with deionised water (2xc3x9710 ml), dried (Na2SO4) and concentrated. Purification by flash silica column chromatography gives Compound M.